Project Summary Chlamydial infections underlie the development of several clinically significant pathologies including blindness, female reproductive tract pathology, cardiovascular diseases, and reactive arthritis. The long term goal of our research is to understand the mechanisms of chlamydial pathogenesis and prevent them. A vaccine against Chlamydia is considered the ideal solution to prevent pathologies in the female upper genital tract (UGT). However, the host immune response, or a subset, is also responsible for these pathologies. An understanding of chlamydial immunopathogenesis is essential to selectively induce protective responses and avoid pathogenic immune response by vaccination, whereas it is not completely understood. To this end, we have previously demonstrated that TNF-? producing CD8+ (CD8) T cells do not contribute significantly to Chlamydia clearance during primary genital tract infection in mice, but mediate UGT pathologies in an antigen (Ag)- specific fashion. The role of CD8 T cells in mediating chlamydial pathology appears to be broadly applicable because we have also validated their contribution to atherosclerosis induction by Chlamydia pneumoniae respiratory infection. The failure of CD8 T cells to aid in chlamydial clearance may be due to down-regulation of MHC class I expression on Chlamydia-infected cell surfaces. Conversely, this suggests that CD8 T cells target uninfected cells in an Ag-specific fashion to induce pathology. Among various cross-presentation mechanisms, the phenomenon of gap junction mediated antigen transport (GMAT) would be the most efficient process to explain this paradigm, as we will explain in this application. The contribution of GMAT to microbial pathogenesis has not been demonstrated. GMAT occurs via channels formed by connexin (CX) proteins, predominantly CX43 (expressed by the gap junction alpha 1 gene, or Gja1). Connexin 43 is expressed on oviduct epithelial cells, and such a mechanism would explain both the pathological effects and low efficiency of chlamydial clearance by CD8 T cells. We have generated mice with a conditional deletion of CX43 in ciliated columnar epithelial cells of the oviduct (described as oveCX43KO in this proposal), and found that connexin 43 mediates Chlamydia-induced UGT pathology. Furthermore, CX43 was engineered into cell lines originally devoid of connexin expression. In a triple cell co-culture model using these cell lines, we have found a role for CX43 in activation of Chlamydia-specific CD8 T cell response. As such, we are uniquely positioned to test our central hypothesis that ?Connexin 43 mediates chlamydial pathogenesis by engaging Ag-specific CD8 T cells?, with these subaims: Aim 1. Determine mechanisms of connexin 43-mediated activation of Chlamydia-specific CD8 T cells, and Aim 2. Determine the role of epithelial connexin 43 in inducing CD8 T cell-mediated oviduct pathology.